This study was carried out from November 2017 to September 2018 in the Faculty of Life Sciences, Ritsumeikan University, Kusatsu city, Shiga prefecture, Japan (34˚58'58.0"N 135˚57'49.2"E).

Black soil (Kanuma Kosan, Tochigi, Japan), vermiculite (Kanuma Kosan), peat moss (Kanuma Kosan), mountain soil (Toyo company, Aichi, Japan), wood chip 1 (particle size 1 cm; DaikenKogyo company, Osaka, Japan), and wood chip 2 (particle size 0.5 cm; DaikenKogyo company, Osaka, Japan) were used for the base soil. Cow manure (Taniguchi Bokujo company, Shiga, Japan), horse manure from a horse ranch (Shiga, Japan), chicken manure from a chicken farm (Shiga, Japan), oil cake (JoY Agris company, Tokyo, Japan), soybean meal (Tamagoya company, Ibaraki, Japan), and bone meal (Tachikawa Heiwa Noen company, Tochigi, Japan) were used as the additive materials. The base soils and additive materials were air dried for 1 week, and then sieved through a 2-mm sieve. The chemical soil (Hanachanbaiyodo company, Nagoya, Japan), which is amended with chemical fertilizer, was considered as a control treatment.

Total carbon (TC) was analyzed using a total organic carbon analyzer (SSM-5000A, Shimadzu, Kyoto, Japan). Total nitrogen (TN), total phosphorus (TP), and total potassium (TK) contents were analyzed by extracting soil samples using the Kjeldahl digestion method followed by analysis using the indophenol blue method, molybdenum blue method, and atomic absorption spectrophotometry, respectively [15] [16]. The total bacterial biomass of the soil was analyzed by quantifying the environmental DNA (eDNA) extracted by the slow-stirring method [17]. Nitrogen (N) circulation and phosphorus (P) circulation activities were examined according to our previous studies [13] [18]. The maximum water holding capacity (WHC) and bulk density were measured by the standard methods [19]. Soil pH (1:2.5 soil-to-water suspension, w/v) was analyzed using a pH meter (LAQUA. F-71, Horiba, Kyoto, Japan).

The base soils and additive materials were dried at 37˚C for 1 week, and then these materials were sieved through a 2-mm sieve. Seven organic soils were prepared by blending the base soils and additive materials. To activate the microbial activities, 200 g of each organic soil was preincubated in a 400 ml pot and maintained 30% of water content for 1 week. The soil sample of each organic soil treatment was subsequently collected for SOFIX analysis [13]. The bacterial biomass was measured on days 0, 3, 5, and 7, while the other parameters were measured on days 0 and 7. The bacterial diversity of the different lots of ideal standard organic soil and the different lots of chemical soils was analyzed on day 0 with polymerase chain reaction denaturing gradient gel electrophoresis (PCR-DGGE) analysis. The organic soils were incubated in the plant factory with 12 h of light: 12 h of dark at 23˚C throughout the experimental period.

Seven organic soils and the chemical soil (control) were used for plant cultivation experiment. A 2 L soil sample was put into a Wagner pot (1/5000a, Fujimoto Kagaku Kogyo company, Tokyo, Japan), and then preincubated at 30% of water content. Brassica rapa var. peruviridis (Komatsuna) seeds were sown in a nursery tray for 1 week, and four seedlings were then transplanted to each Wagner pot. After 4 weeks of cultivation, B. rapa of each treatment were harvested and measured fresh weight, shoot length, root length, chlorophyll content, and the number of leaves. The parameters of B. rapa growth were determined using one-way analysis of variance (ANOVA).The leaf chlorophyll was analyzed by a chlorophyll meter (SPAD-502, Minolta, Tokyo, Japan) and described by SPAD reading values. The experiments were conducted in the plant factory (12 h of light and 12 h of dark; 23˚C).

A best organic standard soil (among seven organic soils) and the chemical soil (base soils + chemical fertilizer) were used for PCR-DGGE analysis. The 16S rRNA bacterial gene was amplified using primers DGGE-F (5’-CGCCC GCCGC GCCCC GCGCC CGTCC CGCCG CCCCC GCCCG CCTAC GGGAG GCAGC AG-3’) and DGGE-R (5’-CCGTC AATTC CTTTG AGTTT-3’) [20]. The amplification reaction was carried out in a 50 μL PCR mixture containing 0.01 ng/μL of DNA template, 1.5 U rTaq DNA polymerase, 5.0 μL of 10× buffer, 5.0 μl of 2 mM dNTPs, 3.0 μl of MgCl₂, and 2.0 μL of 10 mmol/L of each primer. DNA polymerase, dNTPs, and PCR buffer were purchased from TOYOBO (Osaka, Japan), and all primers were synthesized by Sigma-Aldrich (Tokyo, Japan). The thermal PCR profile was as follows: initial denaturation at 95˚C for 1 min, followed by 35 cycles of denaturation at 95˚C for 1 min, primer annealing at 55˚C for 30 s, and extension at 72˚C for 1 min and then a final extension at 72˚C for 5 min. Finally, the amplified 16S rRNA bacterial genes were used for denaturing gradient gel electrophoresis (DGGE) analysis.

DGGE was performed using the D Code System (BioRad Laboratories Inc., California, USA). A total of 20 μl of PCR product was loaded into 8% (w/v) poly acrylamide gel with a denaturant gradient of 27.5% - 67.5%. The gel was then run in 1 × Tris-acetate EDTA buffer at a constant voltage of 70 V at 60˚C for 15 h. The gel was stained using ethidium bromide for 30 min, then rinsed with distilled water. Cluster analysis of the DGGE band pattern was subsequently conducted using the FPQuest Bioinformatics Software (BioRad Laboratories Inc., California, USA).

Base soils and additive materials were selected to construct suitable chemical, physical, and biological characteristics in the organic standard soil. The properties of candidates for the base soil (mountain soil, black soil, peat moss, vermiculite, and wood chips) were measured (Table 1 and Table 2). The TC contents of peat moss and wood chips were higher than those of the other candidate base soils, while the TN and the TP contents of all candidates were low. The maximum WHC of black soil, vermiculite, and wood chips were relatively high but the bulk density of vermiculite, peat moss, and wood chips were low. The components

N.D. = not detected.

difference sizes of wood chips (wood chips 1 and wood chips 2) were almost the same but the bacterial biomass of a wood chip 2 was higher than that of a wood chip 1.

The total nitrogen contents of oil cake, soybean meal, bone meal, chicken manure, and cow manure were above 20,000 mg/kg. The TP contents of oil cake, bone meal, chicken manure, and cow manure were high. The bacterial biomass of all manures was above 6 × 10⁸ cells/g. Among the three types of manure, cow manure was selected because of a well-balanced nutrient content and high bacterial biomass.

The candidates of a standard soil based on SOFIX recommended values (Table 3) were prepared to construct a stable and reproducible organic standard soil. Seven candidates of the organic standard soil were prepared using the base soils and additive materials at different ratios (Table 4 and Table 5). Cow manure, oil cake, soybean meal, and bone meal were added in base soil at 5%, 0.25%, 0.25%, and 0.05% w/w, respectively.

Chemical and physical properties of the seven prepared organic standard soils are shown in Table 6. The TC, TN, TP, and TK contents, and the C/N and C/P ratios of the seven candidate standard soils were 24,000 - 34,740 mg/kg, 1580 - 1840 mg/kg, 1040 - 1160 mg/kg, and 6450 - 9660 mg/kg, and 14 - 20 and 22 - 31, respectively. The bulk density and the WHC of the seven organic standard soils were above 0.5 g/cm³ and 1200 ml/kg, respectively. The chemical and physical properties of the seven organic soils were around SOFIX recommended values. Among seven organic soils, T7 was showed the lowest bulk density but the highest WHC.

The biological properties of the seven candidate organic soils after controlling the water (30% of water content) for 1 week are shown in Figure 1 and Table 7. The bacterial biomass of all candidate organic soils exceeded 6 × 10⁸ cells/g-soil on day 3, and the bacterial biomass of T2, T3, T4, T5, T6, and T7 was greater than 11 × 10⁸ cells/g-soil on day 7. This result indicates that the wood chips increase the bacterial biomass. Among the seven organic soils, T7 showed the highest value of the bacterial biomass. The nitrogen and phosphorus circulation activities of the seven candidates of the organic soil were close to the SOFIX recommended values.

To compare the plant growth, B. rapa cultivation experiment was conducted (Table 8). The performance of Brassica rapa in the seven organic soils was similar

*TC was determined without wood.

Means followed by the same letter do not significantly differ (p < 0.05). Value followed by ± is standard deviation.

or better than that in the chemical soil. An increase of wood chip 2 led to a higher fresh weight and shoot length of B. rapa than that in the chemical soil and in the organic soils with wood chip 1. Especially, B. rapa growth in the organic soil T7 containing 70% (v/v) of wood chip 2 was the highest. These findings suggest that wood chip 2 is the most suitable for B. rapa cultivation. Chlorophyll of plants in the chemical soil used was 19% - 29% higher than those in the organic soils, suggesting that the inorganic nitrogen in the chemical soil was richer than that in the organic soil.

As a result, the organic soil T7 was identified as the best organic standard soil. In the next experiment, comparison of the bacterial diversity between the organic standard soil (T7) and the chemical soil was conducted.

The comparison of the bacterial diversity between the organic standard soil (T7) and the chemical soil were conducted in this study. The bacterial diversities of different lots of the organic standard soil and different lots of the chemical soil were compared (Figure 2). The bacterial diversities of the organic standard soil and the chemical soil were different, even though the same base soil was used in the organic standard soil and the chemical soil. The bacterial diversities of the organic standard soil were similar, but those of different lots of the chemical soil were unstable. The number of bacterial species in the organic standard soil was higher than that in the chemical soil. The organic standard soil was controlled not only by the bacterial biomass but also by the bacterial diversity, suggesting that the bacteria biomass and bacterial diversity seem to be a positive relationship.